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Abstract

Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood.

Objective: To investigate the regulation, and role, of MV biogenesis in VSMC calcification.

Methods and Results: Alexa488-labelled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system and exocytosed from multivesicular bodies (MVBs) via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63 and CD81 and their release was regulated by sphingomyelin phosphodiesterase 3 (SMPD3). Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins.
Elevated extracellular calcium was found to induce SMPD3 expression and the secretion of calcifying exosomes from VSMCs in vitro and chemical inhibition of SMPD3 prevented VSMC calcification. In vivo, MVBs containing exosomes were observed in vessels from chronic kidney disease (CKD) patients on dialysis and CD63 was found to co-localize with calcification. Importantly, factors such as TNF-
and PDGF-BB were also found to increase exosome production leading to increased calcification of VSMCs in response to calcifying conditions.

Conclusions: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.